Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a progressive neurodegenerative disease characterized by tremor with ataxia, brain atrophy and cognitive defects. FXTAS is caused by CGG expansions, varying in the length from ~55 to 200 repeats (CGG55-200), in the 5' UTR of the FMR1 gene. The CGG expansions of more than 200 repeats in the same gene cause the Fragile X Syndrome (FXS), an intellectual disability, distinct from FXTAS. Long CGG expansions silence the transcription of the FMR1 gene; while short expansions elevate the transcription of the FMR1. Numerous models show that the FMR1 mRNA with CGG55-200 repeats or RNA CGG55-200 repeats outside of the FMR1 mRNA cause neurodegeneration. The main pathologic feature of FXTAS is the formation of the ubiquitin (Ub)-positive inclusions that contain the proteasome, RNA-binding proteins and proteins associated with stress response. The role of these inclusions in FXTAS pathogenesis is not well understood. We have examined the activity of the ubiquitin-proteasome system (UPS) in the FXTAS mouse model, rCGG ki mice, and found accumulation of the Ub-protein conjugates in the cerebellum in these mice. We also observed that rCGG repeats increase the levels of un-phosphorylated at S302 form of RNA-binding protein, CUGBP1, named CUGBP1REP. One of the known functions of CUGBP1REP is inhibition of translation of mRNAs by recruiting them into Stress Granules. We generated a mouse model, CUGBP1-S302A knock in mice, which express only CUGBP1REP. We found that S302A ki mice develop brain atrophy at 6-months of age with the loss of the Purkinje cells. The brain atrophy in the S302A ki mice is accompanied by the accumulation of the Ub-proteins in cerebellum showing that CUGBP1REP inhibits the UPS. Since CUGBP1REP is increased in the cerebellum of rCGG ki mice, we suggest that the rCGG repeats inhibit the UPS, at least in part, through the elevation of CUGBP1REP. Based on these data, we propose a hypothesis that rCGG repeats trigger FXTAS pathology by the increase of CUGBP1REP at early stages and that this increase leads to inhibition of the UPS at late stages of the disease contributing to brain pathology. This hypothesis will be tested in the tet-regulated cell lines expressing rCGG repeats (Aim 1) and in rCGG ki, rCGG transgenic and in S302A ki mice (Aim 2). We will determine if the increase of CUGBP1REP precedes the inhibition of the UPS and formation of the Ub- positive inclusions. The identification of the early toxic events which trigger progression of FXTAS will provide a basis for the development of the therapeutic approaches for FXTAS. PUBLIC HEALTH RELEVANCE: The Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a progressive neurological disease affecting elderly patients that carry the CGG expansions in the pre-mutation range in the gene for Fragile X syndrome. There is no cure for this disease. Whereas the current evidence suggests the toxic role of the RNA CGG repeats, the molecular mechanisms by which the rCGG repeats cause brain atrophy in FXTAS are not known. We propose to apply basic science expertise to identify early molecular abnormalities caused by rCGG repeats. The results of this work will provide a basis for the development of the therapy for FXTAS.